Biomedical subjects

Anthony I Magee

Publications and source records attributed to Anthony I Magee.

14 recordsLinked to original sources

PubMed2007

Ras/ERK1/2-mediated STAT3 Ser727 phosphorylation by familial medullary thyroid carcinoma-associated RET mutants induces full activation of STAT3 and is required for c-fos promoter activation, cell mitogenicity, and transformation.

The precise role of STAT3 Ser(727) phosphorylation in RET-mediated cell transformation and oncogenesis is not well understood. In this study, we have shown that familial medullary thyroid carcinoma (FMTC) mutants RET(Y791F) and RET(S891A) induced, in addition to Tyr(705) phosphorylation, constitutive STAT3 Ser(727) phosphorylation. Using inhibitors and dominant negative constructs, we have demonstrated that RET(Y791F) and RET(S891A) induce STAT3 Ser(727) phosphorylation via a canonical Ras/ERK1/2 pathway and that integration of the Ras/ERK1/2/ELK-1 and STAT3 pathways was required for up-regulation of the c-fos promoter by FMTC-RET. Moreover, inhibition of ERK1/2 had a more severe effect on cell proliferation and cell phenotype in HEK293 cells expressing RET(S891A) compared with control and RET(WT)-transfected cells. The transforming activity of RET(Y791F) and RET(S891A) in NIH-3T3 cells was also inhibited by U0126, indicating a role of the ERK1/2 pathway in RET-mediated transformation. To investigate the biological significance of Ras/ERK1/2-induced STAT3 Ser(727) phosphorylation for cell proliferation and transformation, N-Ras-transformed NIH-3T3 cells were employed. These cells displayed elevated levels of activated ERK1/2 and Ser(727)-phosphorylated STAT3, which were inhibited by treatment with U0126. Importantly, overexpression of STAT3, in which the Ser(727) was mutated into Ala (STAT3(S727A)), rescued the transformed phenotype of N-Ras-transformed cells. Immunohistochemistry in tumor samples from FMTC patients showed strong nuclear staining of phosphorylated ERK1/2 and Ser(727) STAT3. These data show that FMTC-RET mutants activate a Ras/ERK1/2/STAT3 Ser(727) pathway, which plays an important role in cell mitogenicity and transformation.

Animals↗

PubMed2006

Rab GTPases containing a CAAX motif are processed post-geranylgeranylation by proteolysis and methylation.

Post-translational modification by protein prenylation is required for membrane targeting and biological function of monomeric GTPases. Ras and Rho proteins possess a C-terminal CAAX motif (C is cysteine, A is usually an aliphatic residue, and X is any amino acid), in which the cysteine is prenylated, followed by proteolytic cleavage of the AAX peptide and carboxyl methylation by the Rce1 CAAX protease and Icmt methyltransferase, respectively. Rab GTPases usually undergo double geranylgeranylation within CC or CXC motifs. However, very little is known about processing and membrane targeting of Rabs that naturally contain a CAAX motif. We show here that a variety of Rab-CAAX proteins undergo carboxyl methylation, both in vitro and in vivo, with one exception. Rab38(CAKS) is not methylated in vivo, presumably because of the inhibitory action of the lysine residue within the AAX motif for cleavage by Rce1. Unlike farnesylated Ras proteins, we observed no targeting defects of overexpressed Rab-CAAX proteins in cells deficient in Rce1 or Icmt, as reported for geranylgeranylated Rho proteins. However, endogenous geranylgeranylated non-methylated Rab-CAAX and Rab-CXC proteins were significantly redistributed to the cytosol at steady-state levels and redistribution correlates with higher affinity of RabGDI for non-methylated Rabs in Icmt-deficient cells. Our data suggest a role for methylation in Rab function by regulating the cycle of Rab membrane recruitment and retrieval. Our findings also imply that those Rabs that undergo post-prenylation processing follow an indirect targeting pathway requiring initial endoplasmic reticulum membrane association prior to specific organelle targeting.

Alkyl and Aryl Transferases↗

PubMed2006

Single-molecule diffusion reveals similar mobility for the Lck, H-ras, and K-ras membrane anchors.

Recent evidence on the occurrence of small (5-700 nm diameter) lipid microdomains in the exoplasmic leaflet of the plasma membrane has evoked interest in the possibility that similar domains may also be present in the cytoplasmic leaflet of the plasma membrane. However, current knowledge about these "lipid rafts", in live cells is limited. One way to obtain insight into the occurrence and the size of lipid rafts is the use of single-molecule microscopy, which allows one to study the diffusive motion of individual molecules with high positional and temporal accuracy. Using this technique, we compared the diffusion behavior of the Lck membrane anchor, which has a high affinity for lipid rafts, to the diffusion behavior of the K-Ras membrane anchor, which has negligible affinity for rafts and compared the results with those of the H-Ras membrane anchor. Surprisingly, we found only minor differences in the diffusion behavior of the various lipid anchors, indicating that putative cytoplasmic leaflet lipid rafts would have to be small (<137 nm diameter) and do not affect the mobility of membrane-anchored molecules much on timescales up to 60 ms.

3T3 Cells↗

PubMed2006

Fluorescence lifetime imaging provides enhanced contrast when imaging the phase-sensitive dye di-4-ANEPPDHQ in model membranes and live cells.

We apply fluorescence lifetime imaging to the membrane phase-sensing dye di-4-ANEPPDHQ in model membranes and live cells. We show that the 1700 ps lifetime shift between liquid-disordered and liquid-ordered phases offers greater contrast than the 60 nm spectral shift previously reported. Detection of cholesterol-rich membrane microdomains is confirmed by observation of the temperature dependence of membrane order and by cholesterol depletion using methyl-beta-cyclodextrin.

Cell Line↗

PubMed2005

Cold-induced coalescence of T-cell plasma membrane microdomains activates signalling pathways.

The plasma membranes of eukaryotic cells are hypothesised to contain microdomains with distinct lipid and protein composition known as lipid rafts. In T cells, cross-linking of lipid raft components triggers signalling cascades. We show that the T-cell antigen receptor (TCR) and a protein tyrosine kinase, Lck, have a patchy plasma membrane distribution in Jurkat T cells at reduced temperatures, although they have a continuous distribution at physiological temperature (37 degrees C). GM1 displays a patchy distribution at reduced temperature after Triton X-100 extraction. The archetypal non-lipid raft marker, the transferrin receptor, displays a more continuous plasma membrane distribution uncorrelated with that of Lck at 0 degrees C. Cold-induced aggregation of the lipid raft-partitioning proteins is accompanied by increased tyrosine phosphorylation and ERK activation, peaking at 10-20 degrees C. Tyrosine phosphorylation is further greatly increased by ligating the TCR with anti-CD3 at 10-20 degrees C. The tyrosine phosphorylation mainly occurred at the plasma membrane, was dependent on Lck and on the surface expression of the TCR. The activation of tyrosine phosphorylation and ERK by TCR ligation at reduced temperature also occurred in human primary T cells. These results support the concept that lipid rafts can form in membranes of live cells and that their coalescence stimulates signalling.

Blotting, Western↗

PubMed2005

A Salmonella typhimurium effector protein SifA is modified by host cell prenylation and S-acylation machinery.

SifA is a Salmonella effector protein that is required for maintenance of the vacuolar membrane that surrounds replicating bacteria. It associates with the Salmonella-containing vacuole but how it interacts with the membrane is unknown. Here we show by immunofluorescence, S100 fractionation and Triton X-114 partitioning that the membrane association and targeting properties of SifA are influenced by a motif encoded within the C-terminal six amino acids. This sequence shares homology with both CAAX and Rab geranylgeranyl transferase prenylation motifs. We characterized the post-translational processing of SifA and showed that the cysteine residue within the CAAX motif is modified by isoprenoid addition through the action of protein geranylgeranyl transferase I. SifA was additionally modified by S-acylation of an adjacent cysteine residue. Similar modifications to host cell proteins regulate numerous functions including protein targeting, membrane association, protein-protein interaction, and signal transduction. This is the only known example of a bacterial effector protein that is modified both by mammalian cell S-acylation and prenylation machinery.

Acylation↗

PubMed2005

Transduction of naive CD4 T cells with kinase-deficient Lck-HIV-Tat fusion protein dampens T cell activation and provokes a switch to regulatory function.

We show here that T cell differentiation can be altered by exposing naive mouse CD4 T cells to altered T cell receptor signaling, achieved by transducing them with a fusion protein consisting of a modified Lck protein lacking the kinase domain and the HIV-Tat protein transduction domain. The Lck-HIV-Tat fusion protein is internalized into naive mouse T cells within 30 min after application to the medium. Activation of transduced cells in vitro resulted in strongly reduced intracellular calcium mobilization, alterations in cytokine profile, and sustained up-regulation of CD25. The cells had suppressive activity in vitro, but no Foxp3 expression. Our data indicate that signals encountered by a naive T cell during its initial activation can profoundly influence its subsequent functional behavior and elicit T cells, which can have regulatory activity.

Animals↗

PubMed2004

Cell surface organization of stress-inducible proteins ULBP and MICA that stimulate human NK cells and T cells via NKG2D.

Cell surface proteins major histocompatibility complex (MHC) class I-related chain A (MICA) and UL16-binding proteins (ULBP) 1, 2, and 3 are up-regulated upon infection or tumor transformation and can activate human natural killer (NK) cells. Patches of cross-linked raft resident ganglioside GM1 colocalized with ULBP1, 2, 3, or MICA, but not CD45. Thus, ULBPs and MICA are expressed in lipid rafts at the cell surface. Western blotting revealed that glycosylphosphatidylinositol (GPI)-anchored ULBP3 but not transmembrane MICA, MHC class I protein, or transferrin receptor, accumulated in detergent-resistant membranes containing GM1. Thus, MICA may have a weaker association with lipid rafts than ULBP3, yet both proteins accumulate at an activating human NK cell immune synapse. Target cell lipid rafts marked by green fluorescent protein-tagged GPI also accumulate with ULBP3 at some synapses. Electron microscopy reveals constitutive clusters of ULBP at the cell surface. Regarding a specific molecular basis for the organization of these proteins, ULBP1, 2, and 3 and MICA are lipid modified. ULBP1, 2, and 3 are GPI anchored, and we demonstrate here that MICA is S-acylated. Finally, expression of a truncated form of MICA that lacks the putative site for S-acylation and the cytoplasmic tail can be expressed at the cell surface, but is unable to activate NK cells.

Base Sequence↗

PubMed2004

Control of immune responses by trafficking cell surface proteins, vesicles and lipid rafts to and from the immunological synapse.

Supramolecular clusters at the immunological synapse provide a mechanism for structuring complex communication networks between cells of the immune system. Regulating intra- and intercellular trafficking of proteins and lipids to and from the immunological synapse provides an additional level of complexity in determining the functional outcome of immune cell interactions. An emergent principle is that molecules requiring tightly regulated cell surface expression, e.g. negative regulators of cell activation or molecules promoting cytotoxicity, are trafficked to the immunological synapse from intracellular secretory as required lysosomes. Many molecules required for the early stages of the intercellular communication are already present at the cell surface, sometimes in lipid rafts, and are rapidly translocated laterally to the intercellular contact. Our understanding of these events critically depends on utilizing appropriate technologies for probing supramolecular recognition in live cells. Thus, we also present here a critical discussion of the technologies used to study lipid rafts and, more broadly, a map of the spatial and temporal dimensions covered by current live cell physical techniques, highlighting where advances are needed to exceed current spatial and temporal boundaries.

Animals↗

PubMed2003

Are prenyl groups on proteins sticky fingers or greasy handles?

This Commentary discusses the work of Dietrich et al. in this issue of the Biochemical Journal, which sheds new light on the biological roles of protein-bound prenyl groups by providing evidence that the alpha-subunit of the heterotrimeric G-protein transducin has a binding site for the geranylgeranyl group of the gamma-subunit.

Animals↗

PubMed2003

Detergent-resistant membranes and the protein composition of lipid rafts.

The plasma membrane of eukaryotic cells contains lipid rafts with protein and lipid compositions differing from the bulk plasma membrane. Several recent proteomic studies have addressed the composition of lipid rafts, but the different definitions used for lipid rafts need scrutinizing before results can be evaluated.

Humans↗

PubMed2003

Imaging metabolism of phosphatidylinositol 4,5-bisphosphate in T-cell GM1-enriched domains containing Ras proteins.

Phosphatidylinositol 4,5-bisphosphate (PI(4,5)P(2)) and Ras proteins are involved in signalling pathways originating at the plasma membrane. The localisation and metabolism of PI(4,5)P(2) was studied in Jurkat T cells using fluorescence microscopic imaging with EGFP-tagged and antibody probes. Software was developed to objectively quantitate colocalisation and was used to show that plasma membrane PI(4,5)P(2) was enriched in lipid raft-containing patches of GM1 ganglioside, formed by crosslinking cholera toxin B-subunit (CT-B). The PI(4,5)P(2) metabolites phosphatidylinositol 3,4,5-trisphosphate and diacylglycerol appeared in plasma membrane CT-B-GM1 patches upon induction of signalling. Transferrin receptor and the CD45 tyrosine phosphatase did not colocalise with CT-B-GM1 patches, whereas the tyrosine kinase Lck, the scaffolding protein LAT, and endogenous Ras proteins did partially colocalise with CT-B-GM1 patches as did transfected EGFP-K-Ras(4B) and EGFP-H-Ras. The results demonstrate that T-cell PI(4,5)P(2) metabolism is occurring in GM1-enriched domains and that Ras proteins are present in these domains in vivo.

Binding Sites↗

PubMed2002

Molecular interactions between desmosomal cadherins.

Desmocollins (Dscs) and desmogleins (Dsgs) are cell-adhesion molecules involved in the formation of desmosome cell-cell junctions and share structural similarities to classical cadherins such as E-cadherin. In order to identify and provide quantitative information on the types of protein-protein interactions displayed by the type 2 isoforms and investigate the role of Ca(2+) in this process, we have developed an Escherichia coli expression system to generate recombinant proteins containing the first two extracellular domains, namely Dsg2(1-2) and Dsc2(1-2). Analytical ultracentrifugation, chemical cross-linking, CD, fluorescence and BIAcore have been used to provide the first direct evidence of Ca(2+) binding to desmosomal cadherins. These studies suggest that Dsc2(1-2) not only exhibits homophilic interactions in solution, but can also form heterophilic interactions with Dsg2(1-2). The latter, on the other hand, shows much weaker homophilic association. Our results further demonstrate that heterophilic interactions are Ca(2+)-dependent, whereas the Ca(2+)-dependence of homophilic association is less clear. Our data indicate that the functional properties of Dsc2(1-2) are more similar to those of classical cadherins, consistent with the observation that Dsc shares a higher level of sequence homology with classical cadherins than does Dsg. In addition to corroborating the conclusions of previously reported transfection studies which suggest the formation of lateral heterodimers and homodimers, our results also provide direct quantitative information on the strength of these interactions which are essential for understanding the adhesion mechanism.

Binding Sites↗

PubMed2001

The Lck SH3 domain negatively regulates localization to lipid rafts through an interaction with c-Cbl.

Lck is a member of the Src family of protein-tyrosine kinases and is essential for T cell development and function. Lck is localized to the inner surface of the plasma membrane and partitions into lipid rafts via dual acylation on its N terminus. We have tested the role of Lck binding domains in regulating Lck localization to lipid rafts. A form of Lck containing a point mutation inactivating the SH3 domain (W97ALck) was preferentially localized to lipid rafts compared with wild type or SH2 domain-inactive (R154K) Lck when expressed in Lck-deficient J.CaM1 cells. W97ALck incorporated more of the radioiodinated version of palmitic acid, 16-[(125)I]iodohexadecanoic acid. Overexpression of c-Cbl, a ligand of the Lck SH3 domain, depleted Lck from lipid rafts in Jurkat cells. Additionally, Lck localization to lipid rafts was enhanced in c-Cbl-deficient T cells. The association of Lck with c-Cbl in vivo required a functional SH3 domain. These results suggest a model whereby the SH3 domain negatively regulates basal localization of Lck to lipid rafts via association with c-Cbl.

Animals↗